Fluctuations of intracellular Ca2+ in growth cones have been shown to regulate axon outgrowth and pathfinding. However, the role of specific Ca2+ entry pathways in determining growth cone behaviors is unclear. I have found that Ca2+ influx through stretch-activated channels suppresses axon outgrowth in Xenopus spinal neurons. My preliminary data suggest that accelerated outgrowth caused by blocking stretch-activated channels is due to reduced Ca2+ influx through select channels. However, reduced baseline Ca2+ has not been detected using standard Ca2+ imaging techniques, suggesting that Ca2+ changes may be within local microdomains. In my first aim, I will use both cytosolic and near-membrane Ca2+ indicators to resolve Ca2+ changes in growth cones after application of stretch-activated channel blockers. Imaging studies will be supported with experiments with fast and slow Ca2+ buffers to investigate the signals necessary for acceleration. In my second aim, I will examine how Rho GTPase activity is modulated by select Ca2+ influx pathways. RhoA, Rac1, and Cdc42 regulate the cytoskeleton to affect growth cone motility and guidance. Biosensors that detect active Rho GTPases will be used to assess the regulation of individual Rho GTPases by Ca2+ flux through stretch-activated channels. Dominant negative mutants of Rho GTPases will be used to determine if they are required for Ca2+-mediated changes in outgrowth. A better understanding of the mechanisms underlying neurite outgrowth and axon guidance during development will benefit human health issues, such as spinal neuron regeneration after injury. [unreadable] [unreadable]